羊膜培养液抑制角膜新生血管的实验研究

Suppression of corneal neovascularization by conditioned culture medium of human amniotic membrane

目的 研究羊膜培养液对小鼠角膜新生血管的抑制作用及机制。方法 应用cornealmicropocket方法 ,将含 10 0ng碱性成纤维细胞生长因子 (bFGF)和 12 %hydronpolymer的缓释颗粒植入角膜层间 ,制备小鼠角膜新生血管模型。实验分对照组、羊膜组和去上皮羊膜组 ,每组 10只眼 ,收集各组培养液于模型制作当日起 ,每日滴眼 4次至术后 7d摄片观测角膜新生血管长入情况并处死动物。对脐静脉血管内皮细胞 (HUVEC)进行体外培养 ,并应用Boyden小房技术和荧光结合(CyQUANT)实验分别检测羊膜培养液对HUVEC细胞迁移和细胞增殖的影响。利用酶联免疫吸附(ELISA)法检测各组培养液中金属蛋白酶抑制剂 1(TIMP1)和 2 (TIMP2 )的含量。结果 羊膜培养液明显抑制由bFGF诱发的小鼠角膜新生血管的形成 ,对照组、羊膜组和去上皮羊膜组角膜新生血管面积分别是 (2 4 8± 0 76 )mm2 、(0 6 4± 0 5 2 )mm2 和 (1 96± 0 6 5 )mm2 。羊膜培养液明显抑制血管内皮细胞的迁移和增殖 ,而对照组和去上皮羊膜组对HUVEC细胞迁移无作用。羊膜培养液中TIMP2水平明显增高 ,而TIMP1的含量无变化。结论 羊膜培养液中TIMP1的含量未增加。而羊膜上皮产生或释放大量的TIMP2 ,抑制血管内皮细胞的迁移和增殖 。

Objective To investigate the effects of the conditioned culture medium (CCM) of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice. Methods AM (with epithelium side up) was cultured in EGM basic medium for 3 days, and the CCM was collected. The CCM consisted of three groups: control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were measured in Boyden chambers and by CyQUANT fluorescence binding assay, respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1, TIMP-2) in the CCM were determined by ELISA assay. Results CNV induced by bFGF was significantly suppressed by the CCM of amniotic membrane. When CCM was applied as an eyedrop 4 times a day for 7 days, the area of CNV was (2.48±0.76) mm 2, (0.64±0.52) mm 2 and (1.96±0.65) mm 2 in control, AM and De-AM groups, respectively. The migration and proliferation of HUVEC were strongly inhibited by the CCM of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. A high level of TIMP-2 was found in AM group, but not in De-AM group. There were no differences in the amount of TIMP-1 in medium among these three groups. Conclusions CCM of amniotic membrane significantly suppresses the CNV induced by bFGF. One of the mechanisms of CCM-mediated suppression is that a high level of TIMP-2 protein is secreted or released into the CCM by AM, which can inhibit the migration and growth of vascular endothelial cells.